Stem cells are cells that can both divide and differentiate into diverse specialized cell types and self-renew to produce more stem cells. In mammals, stem cells are found as either embryonic stem cells, which are isolated from the inner cell mass of blastocysts, or adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues.
Unlike all current treatments relying upon surgical intervention or drugs that modulate physiological activities, stem cells provide a replacement for dysfunctional or degenerating tissue. Therefore, the use of stem cells in replacement therapy could dramatically change the prognosis of many currently untreatable diseases, restore function of damaged organs and correct inborn disorders of metabolism and deficiencies. The importance of technologies associated with expansion of stem cells, both of adult and/or embryonic derivation is illustrated by the numerous preclinical and clinical uses of these cells in treatment of a wide range of diseases.
More recent developments have shown that several stem cells within the hematopoietic compartment, including hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) have the capacity to differentiate into cell types outside the immuno-hematopoietic system, creating an opportunity to use these cell types for tissue repair and regeneration in a wide spectrum of degenerative disorders, end organ failure and dysfunction, and possibly replace organ transplants by cellular therapies.
One of the major clinical uses of stem cells is via hematopoietic stem cell transplants (HSCT). In this procedure a number of cells from a donor are transferred to a recipient in aim of reconstituting the recipient's immune and hematopoietic systems. While performing these transplants, it was realized that immune reconstitution is in fact the best therapy for a number of chemotherapy-resistant cancers such as leukemia, lymphoma, multiple myeloma and a number of solid tumors. Beyond its use in oncology, HSCT harbors the potential of curing non-malignant disorders, such as autoimmune diseases (e.g. Diabetes T1, SLE, and Crohns' disease), inborn errors of metabolism and enzyme deficits, hemoglobinopathies or congenital and acquired immunodeficiency. HSCT has been used for non-cancer indications, showing significant positive results. Nevertheless, this procedure is currently employed for life threatening conditions because of its severe toxicity effects of which Graft versus host disease (GvHD) is the most critical.
When using this approach for the treatment of cancer, it was most frequently performed following myeloablative preconditioning by eradication of the immuno-hematopoietic system through aggressive radio-chemotherapy so as to prevent graft rejection. The recent realization that a hosts's immuno-hematopoietic system does not need to be eradicated prior to grafting had been a significant advancement, thus replacing myeloablative preconditioning with non-myeloablative preconditioning and reduced intensity conditioning (RIC). The use of non-myeloablative preconditioning has improved significantly, yet not sufficiently, life-threatening situations caused by vital organ dysfunction, failure of engraftment and intractable infections. Since host's hematopoietic system is not eradicated by RIC, it can recover in case the donor cells fail to engraft. With time, the donor graft takes over, a process that facilitates the generation of graft versus tumor (GvT) and graft versus autoimmunity (GvA) reactions, yet further expose the patient to GvHD critical morbidity and mortality.
The identification of the type 1 transmembrane protein/adhesion molecule, the sialomucin CD34 as a marker of hematopoietic stem cells led to the ability to use CD34+ cell selection as a means of concentrating hematopoietic stem cells for transplantation purposes. CD34 markers is absent from some stem cells and found also on various subtypes of blood precursors. Using such a positive selection method results in loss of some of the beneficial stem cells. Moreover, it yields a mixed cell population of stem and progenitor cells with some later precursor cells. Transplantation of such a mixed cell population decreases transplantation success [Askenasy N. et al., Current Stem Cell Research and Therapy 2006; 1:85-94]. Therefore, a need arose for a selection method which retains all the cells needed for hematopoietic reconstitution while discarding the adverse effects causing cells.
Unlike somatic cells, hematopoietic stem and progenitor cells (HSPC), mesenchymal stromal cells and neural progenitors (NP) have been documented to be insensitive to injury factors such as those inflicted both by radio-chemotherapy and by secondary factors released into the marrow space as a result of massive death of resident hematopoietic cells. HSPC are particularly resistant to apoptotic signals transduced by tumor necrosis factor (TNF) family receptors, which are instead utilized to deliver growth signals in the most primitive progenitors. In murine models, hematopoietic progenitors have been shown to acutely upregulate several TNF family receptors under conditions of injury and stress. In the transplant setting, this physiological mechanism prioritizes more primitive progenitors for engraftment over apoptosis-sensitive donor cells. Therefore, exposure of a transplant population to TNF family apoptosis-inducing ligands such as FasL, Trail, Tweak or TNF-α results in negatively selecting the stem cell population, as cell populations sensitive for TNF-family ligand induced apoptosis undergo apoptosis and are removed from the transplant. The use of such method in a murine model has been disclosed in patent application WO 2007/138597.
The composition of the donor graft is a significant parameter of stem cell transplant. It has been shown that a threshold number of progenitors is required in order to ensure engraftment. In addition, the presence of some non-stem cell subsets substantially improves the probability of engraftment, such that the transplantation of heterogeneous mixtures of cells is more effective than transplantation of purified progenitors. The most significant subsets within the donor graft are (CD4+ and CD8+) T cells as they have been demonstrated to counteract rejection and support hematopoietic progenitor engraftment within the bone marrow microenvironment. However, transplantation of allogeneic T cells into partially immunosuppressed recipients supports durable engraftment, which mediates a potentially lethal graft versus host reaction (GvH) or graft versus host disease (GvHD). Mature donor T cells mediate this reaction, whereas donor T cells that develop de novo after transplantation are tolerant to the host. Extensive efforts have been invested in dissociation between T cell subsets that mediate GvH and support engraftment; however the experimental evidence has been so far inconclusive.
Graft versus host disease (GvHD) includes an acute phase reaction, usually within the first 100 days post-transplantation, and a chronic reaction with more indolent progression but equally detrimental consequences. Importantly, both reactions are triggered by initial inflammation mediated by mature donor T cells within days from transplantation. Acute GvHD is usually treated by immunosuppressive therapy, which has negative effects on hematopoietic reconstitution, whereas there is no current effective therapy for the chronic reaction. The traditional approach to prevention of GvHD consists of depletion of mature T cells from the donor inoculum, at times accompanied by B lymphocytes, using cell surface makers such as CD3, CD4, CD8. Intensive efforts to achieve more selective T cell depletion (TCD) using various cell surface markers have failed.
More specific depletion has been achieved with decent results by elimination of reactive T cell subsets using apoptotic signals following ex vivo sensitization against host antigens. Sensitized T cells express repertoires of activation-related molecules, become sensitive to activation-induced cell death (AICD) and proliferate at fast rates, characteristics that are used for specific depletion. However, there are several major hurdles to this approach to selective elimination. First, sensitization of T cells is a process of repeated exposure to antigens, which requires 3-7 days of ex vivo incubation. Consequently, T cell activation has to be performed at least 3 days prior to transplantation. Second, sensitization and activation that render fast-cycling T cells susceptible to AICD, also induces the development and expansion of effector/memory T cells, whose persistent alloreactivity can initiate and propagate GvHD. Effector/memory T cells are relatively resistant to Fas cross-linking, in part due to inherent low levels of caspase-3, resulting in an apoptosis-resistant phenotype that predisposes patients to acute and chronic GvHD after infusion of the ex vivo cultured T cells. Third, the most effective ex vivo sensitization is against components of the major histocompatibility complex (MHC), the disparity of which leads to dominant alloresponses in the transplant setting. However, the GvHD reaction is stimulated primarily by minor histocompatibility complex antigens (miHA) and targets mainly tissue-specific antigens (TSA). Normal and aberrant tissue epitopes are exposed by injury inflicted by pre-transplant conditioning and by inflammation. An effective method for selective depletion of T cells which induce GvHD, while retaining subsets of cells which support GvT and transplant engraftment, is yet to be found.